World Journal of Microbiology & Biotechnology (2006) 22:745–752 Ó Springer 2006

DOI 10.1007/s11274-005-9100-6

Characterization of selected actinomycetes degrading polyaromatic hydrocarbons

in liquid culture and spiked soil

Leticia Pizzul*, Marı́a del Pilar Castillo and John Stenström

Department of Microbiology, Swedish University of Agricultural Sciences, Box 7025, SE-750 07, Uppsala, Sweden
*Author for correspondence: Tel.: +46-18-673284, Fax: +46-18-673392, E-mail: Leticia.Pizzul@mikrob.slu.se

Received 9 September 2005; accepted 10 November 2005

Keywords: Actinomycetes, biodegradation, biosurfactants, co-substrate, Gordonia, polycyclic aromatic hydrocar-

bons, rapeseed oil, Rhodococcus

Summary

Five strains of the Rhodococcus and Gordonia genera were evaluated for their potential use in bioremediation of
polycyclic aromatic hydrocarbons (PAH) with or without another substrate (co-substrate). Their ability to produce
biosurfactants or to degrade phenanthrene when growing on glucose, hexadecane and rapeseed oil was tested in
liquid medium at 30 °C. All strains showed biosurfactant activity. The highest reduction in surface tension was
recorded in whole cultures of Rhodococcus sp. DSM 44126 (23.1%) and R. erythropolis DSM 1069 (21.1%) grown
on hexadecane and Gordonia sp. APB (20.4%) and R. erythropolis TA57 (18.2%) grown on rapeseed oil. Cultures of
Gordonia sp. APB and G. rubripertincta formed emulsions when grown on rapeseed oil. After 14 days of incubation,
Rhodococcus sp. DSM 44126 degraded phenanthrene (initial concentration 100 lg ml)1) as sole carbon source
(79.4%) and in the presence of hexadecane (80.6%), rapeseed oil (96.8%) and glucose (below the limit of detection).
The other strains degraded less than 20%, and then with a co-substrate only. Rhodococcus sp. DSM 44126 was
selected and its performance evaluated in soil spiked with a mixture of PAH (200 mg kg)1). The effect of the
addition of 0, 0.1 and 1% rapeseed oil as co-substrate was also tested. Inoculation enhanced the degradation of
phenanthrene (55.7% and 95.2% with 0.1% oil and without oil respectively) and of anthracene (29.2% with 0.1%
oil). Approximately 96% of anthracene and 62% of benzo(a)pyrene disappeared from the soil (inoculated and
control) after 14 days and anthraquinone was detected as a metabolite. Rhodococcus sp. DSM 44126 was identified
as Rhodococcus wratislaviensis by 16S rRNA sequencing and was able to degrade anthracene as sole carbon source
in liquid culture.

Introduction priority environmental pollutants (Kanaly & Harayama

Actinomycetes are good candidates for application in
soil bioremediation. They utilize a wide range of carbon
sources, degrade complex polymers such as lignin and
possess some of the advantageous characteristics of
fungi, i.e. mycelial growth, production of spores, resis-
tance to drought and production of extracellular
enzymes (McCarthy & Williams 1992; April et al. 2000).
Many genera have the ability to degrade different
organic pollutants, especially members of the genus
Rhodococcus (Behki et al. 1993; Bouchez et al. 1997;
Dean-Roos et al. 2001; Larkin et al. 2005).
Polycyclic aromatic hydrocarbons (PAH) are organic
pollutants found at sites associated with gasworks,
petroleum, coal tar and wood-preserving industries
(Wilson & Jones 1993). PAH can have mutagenic, tera-
togenic and carcinogenic properties and are classified as
2000). Microorganisms, many of them actinomycetes,
are able to degrade or transform PAH (Cerniglia &
Heitkamp 1990; Wattiau 2002; Wick et al. 2002). Low
molecular weight PAH are frequently used as the sole
source of carbon and energy (Weissenfels et al. 1990;
Wattiau 2002) while mineralization of PAH with more
than three aromatic rings is less common (Walter et al.
1991; Hwang & Cutright 2002) and in some cases
only possible in the presence of another substrate
(co-substrate) (Kanaly & Harayama 2000). The use of
diesel oil enhances degradation of benzo(a)pyrene, most
likely by inducing co-metabolic biotransformation and
by promoting emulsification through the production of
surface-active compounds and microbial proliferation
on the co-substrate (Kanaly et al. 2000).
Most PAH have low water solubility, bind tightly to soil
particles and can be trapped within soil organic matter
746
and soil micropores (Tiehm 1994; Hatzinger & Alexander
1995). This results in a reduction in bioavailability, one of
the most important factors limiting bioremediation (At-
agana et al. 2003). One approach to overcome this prob-
lem is to increase PAH solubility by using biosurfactants
(Barkay et al. 1999). The production of surface-active
molecules has been demonstrated in the presence of sev-
eral compounds with different water solubility, e.g. glu-
cose (Guerra-Santos et al. 1984), n-alkanes (Ramsay et al.
1988, Singer & Finnerty 1990) and oils (Christofi & Iv-
shina 2002). When utilizing alkanes at low temperatures,
Rhodococcus sp. strain Q15 shows physiological cellular
responses, for example production of cell-bound bio-
surfactants and higher cell surface hydrophobicity
(Whyte et al. 1999), while R. erythropolis produces bio-
surfactants when growing on hydrophilic and hydro-
phobic substrates (Pirog et al. 2004).
The objectives of this work were to (i) identify strains
of the group actinomycetes with potential for use in
bioremediation of PAH; (ii) test their capacity to pro-
duce biosurfactants when growing in liquid media with
hydrophilic (glucose) or hydrophobic (hexadecane and
rapeseed oil) substrates as the carbon source; (iii) study
their ability to degrade phenanthrene in liquid culture
with or without the addition of the above-mentioned
substrates; (iv) assess the effect of inoculation with the
most efficient strain on degradation of a mixture of PAH
(phenanthrene, anthracene, pyrene and benzo(a)pyrene)
in a spiked soil with or without rapeseed oil as a co-
substrate; and (v) identify this strain to species level and
test its ability to degrade other PAH as sole carbon
source in liquid culture.
Materials and methods
Microorganisms
The strains used in this study are listed in Table 1. Rho-
dococcus erythropolis TA57 was a gift from Søren
Andersen, GEUS, Denmark. Gordonia sp. APB was a gift
from Janet Jansson, Department of Microbiology,
Swedish University of Agricultural Sciences, Sweden. The
other strains used were obtained from the German Col-
lection of Microorganisms and Cell Cultures (DSMZ).
Chemicals
Anthracene (>96%) and phenanthrene (>97%) were
purchased from Merck (Hohenbrunn, Germany),
Table 1. Strains of actinomycetes screened for phenanthrene degradation and production of biosurfactants.
Strain Isolated from
Gordonia sp. APB Soil enriched with chlorophenol
G. rubripertincta DSM 46038 Soil
Rhodococcus sp. DSM 44126 PAH-contaminated soil
R. erythropolis TA 57 Agricultural soil
R. erythropolis DSM 1069 Agricultural soil
L. Pizzul et al.
pyrene (98%) was purchased from Aldrich, anthraqui-
none (>99%) was supplied by Flucka (Steinheim,
Germany) and benzo(a)pyrene (97%) was supplied by
Sigma-Aldrich (Steinheim, Germany). Toluene (HPLC
grade) was provided by Fisher Scientific (UK) and
hexadecane (>99%) was purchased from Merck-
Schuchardt (Hohenbrunn, Germany). Glucose was
supplied by AnalaRÒ, BDH (Poole, England). Rapeseed
oil was a gift from JTI-Swedish Institute of Agricultural
and Environmental Engineering (Uppsala, Sweden).
Biosurfactant activity with different carbon sources
All the actinomycetes were tested for their potential for
biosurfactant production using hexadecane, rapeseed oil
and glucose as carbon sources. Erlenmeyers (250-ml)
with 200 g of glass beads (5 mm diameter) were auto-
claved for 20 min at 120 °C. Bacteria were first grown in
GYM Streptomyces medium (4 g glucose l)1, 4 g yeast
extract l)1 and 10 g malt extract l)1; pH 7.2) at 30 °C on
a shaker (150 rev min)1) for 3 days. A portion of the
culture was diluted 1:1000 in an autoclaved (20 min,
120 °C) minimal salt medium (MSM) resulting in a
bacterial concentration of 105 c.f.u. ml)1. The MSM was
adapted from Mandelbaum et al. (1993), using 1 g of
KNO3l 1 instead of atrazine. Fifty ml of the medium
were added aseptically to the Erlenmeyers containing
the glass beads and placed on a shaking table at 40
rev min)1. Hexadecane (0.1% v/v), rapeseed oil (0.1%
w/v) or glucose (0.2% w/v) was used as the only carbon
source. Sterile controls for each carbon source were in-
cluded. Triplicates were run for each treatment.
The biosurfactant activity was estimated as the per-
centage decrease in surface tension (ST) and as the
production of emulsions by measuring the emulsifica-
tion index (EI24). Surface tension was measured using a
Educational Tensiometer K6 (Krüss GmbH, Germany)
and the percentage decrease in ST between 24 and 168 h
was estimated. The emulsification activity was measured
in a separate trial by determining the EI24 index at
168 h. The microorganisms were grown as described
above but 1% w/v rapeseed oil was used. Two ml of
sample were mixed with 2 ml of hexane, shaken vigor-
ously for 2 min and left standing for 24 h to allow the
formation of an emulsion at the interface. The per-
centage EI24 was determined as height of the emulsified
layer (mm) divided by total height of the liquid column
 100 according to Iqbal et al. (1995). As the biological
substances that possess surface-active or emulsifying
properties may be free or cell-associated, reduction of
Degrades References
Chlorophenols Pérez-Bercoff 2003
Hydrocarbons from DSMZ homepage
Naphthalene Grund et al. 1992
Triazine Andersen et al. 2001
Lignin, phenol Eggeling & Sahm 1980; Trojanowski et al. 1977
Characterization of PAH degraders
ST and EI24 was measured in the whole culture and in
the supernatant. To obtain a supernatant the culture
liquid was centrifuged at 14784g for 15 min.
Degradation of phenanthrene in liquid culture
by actinomycetes in the presence of hydrophilic
and hydrophobic co-substrates
Twenty grams of glass beads (5 mm diameter) were
added to 50-ml tubes and autoclaved for 20 min at
120 °C. Phenanthrene in acetone was added aseptically
onto the glass beads in order to reach a final concen-
tration in the medium of 100 lg ml)1. The acetone was
allowed to evaporate.
The microorganisms were grown as described above
and 10 ml of the medium containing 105 c.f.u. ml)1 were
added aseptically to the tubes containing phenanthrene.
Glucose (0.2% w/v), hexadecane (0.1% v/v) or
rapeseed oil (0.1% w/v) was used as an additional
carbon source. The tubes were placed on a horizontal
shaking table at 40 rev min)1 with an inclination of
about 10° to the horizontal. The incubation period
was 14 days at 30 °C. Sterile controls for each carbon
source were included. Duplicates were run for each
treatment.
Degradation of PAH by Rhodococcus sp. DSM 44126
in soil
The soil used in the experiment was an agricultural
topsoil (14% clay, 1.8% organic carbon, pHwater 6.6)
collected in Uppsala, Sweden. It was contaminated with
50 mg kg)1 of each of the following PAH: anthracene,
phenanthrene, pyrene and benzo(a)pyrene in an acetone
solution. The soil spiking was carried out according to
Brinch et al. (2002) by treating a sub-sample of the soil
with the PAH solution, mixing thoroughly after the
acetone had evaporated and finally mixing again with
the remaining sample. Rapeseed oil (0.1 and 1% w/w)
was used as co-substrate.
Microorganisms were cultured for 2 days in GYM
Streptomyces medium on a shaker (150 rev min)1) at
30 °C. The culture was diluted once in sterile tapwater
and added to the soil (106 c.f.u. g)1). The soil was
incubated for 14 days at 30 °C and the water content
was kept at 60% of the water-holding capacity. Soil
without the addition of microorganisms was used as the
control and the experiment was carried out in duplicate.
PAH content was measured at the beginning of the
experiment and after 14 days of incubation.
Characterization of Rhodococcus sp. DSM 44126
Rhodococcus sp. DSM 44126 was characterized by the
16S rDNA sequence. Isolation of chromosomal DNA,
PCR and direct sequencing of the PCR products was
carried out following established methods, as described
by Kim et al. (1998). The 16S rRNA gene sequences
were aligned manually, using the AL 16S programme
747
(provided by J. Chun, School of Biological Sciences,
Seoul National University) against corresponding
sequences retrieved from the DDBJ, EMBL and Gen
Bank databases.
The strain was also tested for its ability to degrade
anthracene, pyrene and benzo(a)pyrene as the sole car-
bon and energy source in liquid medium. The same
procedure described for degradation of phenanthrene in
liquid culture was used.
Analytical methods
PAH in soil were extracted by adding 10 ml of toluene
and 10 ml of 0.05 M sodium pyrophosphate to the tubes
containing 10 g soil and shaking vigorously for 16 h on
a shaking table (adapted from Karstensen 1996). The
extracts were centrifuged for 10 min at 492g and a
portion of the toluene layer was cleaned in an alumina
column (IsoluteÒ). PAH in liquid medium were
extracted by adding 10 ml toluene to each tube and
shaking for 1 h. After 10 min centrifugation at 492  g,
a sample of the supernatant was analysed directly by
GC–MS. The recovery from the spiked soil of all the
PAH used was higher than 95%.
GC–MS analysis was performed using a HP 6890
Series GC-system equipped with a HP 5971 Mass
Selective Detector and a HP 19091S-433 capillary col-
umn (0.25 mm inner diameter, 30 m length, 0.25 lm
thickness). The oven programme was 80 °C for 4 min
followed by ramping at 7 °C min)1 up to 310 °C
maintained for 4 min. The injector temperature was
250 °C. Quantification was performed using external
standards. Anthraquinone identification was carried out
using the Wiley 275 mass spectral library.
Results and discussion
The strains used in this work are listed in Table 1.
These strains were chosen based on an initial evalua-
tion of 17 members of the group actinomycetes for
their potential for surfactant production and degra-
dation of aromatic compounds (data not shown).
Special emphasis was placed on members of the gen-
era Gordonia and Rhodococcus, since they are often
found at polluted sites, especially oil- and PAH-con-
taminated soils (Dean-Roos et al. 2002; Lin et al.
2005). They also show high cell hydrophobicity and
release surface-active substances (Lang & Philp 1998;
Nazina et al. 2003) and can degrade several organic
compounds by mechanisms that may be of interest in
PAH degradation. Moreover nocardioform actinomy-
cetes in general may play a crucial role in the degra-
dation or mineralization of PAHs in soils (Kästner
et al. 1994).
The most promizing strains were further studied for
their ability to produce surfactants using rapeseed oil,
glucose and hexadecane as substrates and to degrade
phenanthrene in liquid culture with or without the
748
presence of the above mentioned carbon sources. The
strain showing the best performance was then tested in a
PAH-spiked soil amended with different concentrations
of rapeseed oil.
Biosurfactant activity with different carbon sources
The production of biosurfactants was determined based
on the ability of the strains to decrease the surface ten-
sion (ST) of the liquid medium. All the strains reduced
ST when grown on hexadecane or rapeseed oil as the
carbon source (Figure 1). No decrease in ST in any case
was observed with glucose (Figure 1). These results
confirm that the production of biosurfactants is highly
related to the substrate (Georgiou et al. 1992). The
largest reductions in ST in the period between 24 h and
168 h were measured for Rhodococcus sp. DSM 44126
and R. erythropolis DSM 1069 grown in hexadecane
(23.1 and 21.1% respectively), and Gordonia sp. APB
and R. erythropolis TA57 grown in rapeseed oil (20.4
and 18.2% respectively), in all cases for whole cultures.
Figure 1 also shows that Gordonia sp. APB and
G. rubripertincta were the only strains that formed
detectable emulsions, with EI24 values of 29.9 and
41.1% after 168 h of growth in MSM with rapeseed oil
as the carbon source. Similar values were reported for
G. alkanivorans growing in liquid media supplemented
with kerosene or diesel (Lin et al. 2005). Emulsification
activity was detected only when bacterial cells were
present, indicating that it was related to cell hydropho-
bicity and/or cell-associated substances. This was not
unexpected, since cells of the R (rough) variant of
R. rubropertinctus (synonym G. rubripertincta) had
shown a high hydrophobicity index after mixing with
hexadecane (Mil’ko & Egorov 1994). A decrease in
surface tension in the whole culture and the supernatant
Figure 1. Production of biosurfactants (measured as percentage reduction in surface tension (ST) between 24 and 168 h) by Gordonia and
Rhodococcus strains grown on 0.1% hexadecane and 0.1% rapeseed oil, whole culture (W) and supernatant (S) and emulsification activity (EI24)
grown on rapeseed oil (whole culture). Error bars show the standard deviation (n=3).
L. Pizzul et al.
was also observed for these two strains in the presence of
the vegetable oil (Figure 1), indicating that production
of biosurfactants might also be taking place. These
results were similar to those reported for another Gor-
donia species, G. amarae, where it was concluded that
both cells and an extracellular biosurfactant were in-
volved in the formation and stabilization of foam
(Iwahori et al. 2001).
The Rhodococcus strains were not able to form a
stable emulsion in spite of their surface activity when
grown in rapeseed oil (Figure 1), supporting the finding
that there is no correlation between the ability to reduce
surface tension and the ability to form emulsions
(Willumsen & Karlson 1997).
Degradation of phenanthrene in liquid culture in the
presence of hydrophilic and hydrophobic co-substrates
The degradation of phenanthrene as the sole carbon
source and with rapeseed oil, hexadecane and glucose as
co-substrates is shown in Figure 2. Rhodococcus sp.
DSM 44126 transformed 79.4% of the phenanthrene
alone after 14 days and similar values were obtained in
the presence of hexadecane (80.6%). The presence of
rapeseed oil (0.1%) increased the degradation values up
to 96.8% and with glucose (0.2%) the concentration of
phenanthrene was below the limit of detection. The
same strain has been studied previously for its ability to
grow on naphthalene as the sole carbon and energy
source (Grund et al. 1992) but, to our knowledge,
nothing has previously been reported about its metab-
olism of phenanthrene.
The other strains did not utilize phenanthrene as the
sole carbon source. However, they all degraded phen-
anthrene to different extents when a co-substrate was
Characterization of PAH degraders
Figure 2. Percentage degradation of phenanthrene (100 mg l)1) in liquid culture by Gordonia and Rhodococcus strains with or without
co-substrates after 14 days of incubation at 30 °C. Error bars show the standard deviation (n=3).
present, although never reaching reduction values higher
than 20% (Figure 2).
The enhanced degradation of phenanthrene in the
presence of rapeseed oil might partly be the result of an
increase in its solubility due to the production of
biosurfactants or the emulsification activity in the
presence of this carbon source (Figure 1). However, the
enhancement with glucose as the co-substrate cannot be
explained by biosurfactant activity since no changes in
ST were detected with this carbon source. Co-metabolic
degradation or an increase in microbial growth might
have occurred. This could also be true for the hydro-
phobic co-substrates.
Degradation of PAH in soil by Rhodococcus sp. DSM
44126 in the presence of rapeseed oil
In the first part of this work the experiments were con-
ducted in liquid culture conditions and they showed
that Rhodococcus sp. 44126 was the best phenan-
threne degrader (Figure 2) and had potential as a bio-
surfactant producer (Figure 1). Rapeseed oil showed a
potential for inducing biosurfactant or emulsifying
activity (Figure 1) and enhanced phenanthrene degra-
dation (Figure 2). The addition of vegetable oils
enhanced the solubility and removal of PAH from soil
(Berg Schuur & Mattiasson 2003, Bogan et al. 2003) and
e.g. peanut oil has been suggested as a natural,
non-toxic, cost-effective and biodegradable extracting
agent (Pannu et al. 2003). From these results we decided
to test the effect of inoculation and addition of different
concentrations of rapeseed oil on the degradation of
PAH in soil spiked with a mixture of anthracene,
phenanthrene, pyrene and benzo(a)pyrene.
The results for the individual PAH are presented in
Table 2. The inoculation with Rhodococcus sp. DSM
44126 enhanced the degradation of phenanthrene but
749
unlike what was observed in liquid culture, the presence
of oil did not favour the utilization of phenanthrene. In
the soil, degradation of phenanthrene decreased with the
increase of co-substrate, while with the addition of 1%
rapeseed oil the values did not differ from the
non-inoculated control. This may be explained by a
preference for the co-substrate over the hydrocarbon,
which could eventually be degraded after the utilization
of the oil. The degradation values in the non-inoculated
soil suggested a high activity of the indigenous microfl-
ora and the same repressive effect of the oil.
The levels of pyrene did not differ between the treat-
ments after 14 days (Table 2). About 20% disappeared
in all cases, probably due to the native microorganisms.
The addition of 0.1% rapeseed oil slightly stimulated
the degradation of anthracene in the inoculated soil
compared to the control. This level of rapeseed oil did
not affect the degradation of benzo(a)pyrene. These two
compounds showed similar behaviour for the other two
levels of co-substrate. Inoculation with Rhodococcus sp.
DSM 44126 without addition of rapeseed oil did not
affect degradation but after 2 weeks the levels decreased
by approximately 30% for anthracene and 10% for
benzo(a)pyrene. The most important differences were
found in the soil treated with 1% oil. Decreases of 93
and 64% in anthracene and benzo(a)pyrene respectively
were observed after only 14 days in both inoculated and
non-inoculated soil. These decreases coincided with the
appearance of a new peak in the chromatograms. Using
the Wiley 275 mass spectral library, this compound was
identified as the anthracene degradation product
anthraquinone, with a match quality of 96%. Its identity
was also confirmed by running a standard, which
showed the same retention time as that observed for the
soil samples. Anthraquinone has been reported as one of
the major metabolites of anthracene by white rot fungi
(Bezalel et al. 1996, Cajthaml et al. 2002) and
750 L. Pizzul et al.
Table 2. Effect of inoculation with Rhodococcus sp. DSM 44126 on the degradation of PAH after 14 days of incubation in liquid culture and soil,
with or without the addition of rapeseed oil.

Degradation (%)a

PAH mixture in soil PAH mixture in liquid medium

Oil content (%) Inoculated Control Inoculated Control

Phenanthrene 0 95.2 ± 1.5* 71.9 ± 6.5 41.8 ± 10.2 –c

0.1 55.7 ± 6.4* 31.9 ± 1.8 ndb nd
0.5 nd nd 76.6 ± 13.1 –
1 22.6 ± 4.0 32.5 ± 18.5 nd nd
Anthracene 0 37.2 ± 5.5 28.7 ± 4.71 – –
0.1 29.2 ± 0.3* 25.9 ± 0.54 nd nd
0.5 nd nd 46.0 ± 3.9 39.0 ± 12.1
1 97.0 ± 1.7 95.1 ± 2.16 nd nd
Pyrene 0 29.7 ± 7.3 18.5 ± 10.1 – –
0.1 20.8 ± 2.0 20.2 ± 0.5 nd nd
0.5 nd nd – –
1 26.9 ± 8.0 22.3 ± 2.2 nd nd
Benzo(a)pyrene 0 9.2 ± 0.2 10.6 ± 0.7 – –
0.1 13.5 ± 1.6 13.7 ± 1.3 nd nd
0.5 nd nd 26.2 ± 2.5 33.3 ± 5.5
1 61.2 ± 4.0 63.7 ± 4.8 nd nd
a
The values are means ± standard deviations in duplicate samples. Degradation values for inoculated and control soil are compared within
rapeseed oil levels.
b
nd, not determined.
c
–, no degradation.
*
significant at the 5% level.

9,10-anthraquinone has been found as a dead-end
product in the degradation of anthracene by Mycobac-
terium sp. PYR-1 (Moody et al. 2001). In addition to
biological degradation, anthraquinone can be formed by
chemical and photooxidation processes (Kraus et al.
1999, Mallakin et al. 2000). No degradation products
from benzo(a)pyrene could be identified.
The degradation of the mixture of PAH in the pres-
ence of 0.5% rapeseed oil was then tested in liquid
medium, sterile or inoculated with Rhodococcus sp.
DSM 44126, following the same procedure used for the
evaluation of phenanthrene degradation with or without
different co-substrates. Degradation of phenanthrene
was observed only in the inoculated samples and it was
stimulated by the presence of oil (Table 2). No changes
in anthracene or benzo(a)pyrene were observed when
only PAH were added to the medium. However, trans-
formations occurred whenever rapeseed oil was present
in both sterile and inoculated samples, higher for
anthracene than for benzo(a)pyrene (Table 2), and in all
the cases were accompanied by the formation of
anthraquinone (data not shown). From these results it
could be concluded that in addition to biological
transformation, other processes were involved in the
disappearance of anthracene and benzo(a)pyrene. A
combination of co-metabolic processes, increased bio-
availability and non-biological chemical reactions, all
due to the addition of the rapeseed oil, could be
responsible for the transformation of anthracene and
benzo(a)pyrene in soil but on the basis of our observa-
tions it was not possible to differentiate between these
mechanisms.
Characterization of Rhodococcus sp. DSM 44126
Rhodococcus sp. DSM 44126 was isolated from a soil
sample contaminated with PAH and has been studied
for the catabolic pathways of naphthalene degradation
(Grund et al. 1992). Our study showed that this strain
was also efficient in degrading phenanthrene in liquid
medium, both as a sole carbon and energy source and in
the presence of glucose, hexadecane and rapeseed oil as
co-substrates. Moreover, its ability to degrade phenan-
threne was observed in spiked soil. The strain was fur-
ther tested for degradation of phenanthrene, anthracene,
pyrene or benzo(a)pyrene as the sole carbon source in
liquid medium and the results revealed that it is also able
to transform anthracene (30% in 14 days). This finding
confirmed the tendency observed in the results of
anthracene degradation in the spiked soil for the treat-
ments without addition of oil (Table 2).
The complete 16S rDNA nucleotide sequence of
Rhodococcus sp. DSM 44126 showed 100% similarity
with R. wratislaviensis. The ability of R. wratislaviensis
to degrade nitroaromatic compounds has been reported
previously (Gemini et al. 2005, Navrátilová et al. 2005)
but not its ability to degrade phenanthrene and
anthracene.
Conclusions
The presence of a co-substrate in liquid medium stim-
ulated the degradation of phenanthrene by all the
strains tested, especially by Rhodococcus sp. DSM
Characterization of PAH degraders
44126. In the case of the hydrophobic co-substrates,
this could partly be due to enhancement of phenan-
threne solubility by the production of biosurfactants or
emulsifiers.
Rhodococcus sp. DSM 44126, identified as R. wrati-
slaviensis, was a good phenanthrene degrader, both in
liquid medium and in spiked soil. It was also able to
degrade anthracene in liquid medium and to a lesser
extent in soil. A longer incubation time would be needed
to follow the degradation of other PAH and the influ-
ence of the co-substrate.
The emulsification ability observed for Gordonia sp.
APB and G. rubripertincta could be useful for the
enhancement PAH bioavailability in soil. Gordonia sp.
APB has the additional advantage of being a degrader
of phenol, a compound that it is often found in com-
bination with PAH in creosote-contaminated sites.
Inoculation with a co-culture of this strain and Rhodo-
coccus wratislaviensis could be tested for cleaning up
such contaminated soils.
The addition of 1% rapeseed oil to the soil spiked
with a mixture of PAH had a very positive effect in
reducing anthracene and benzo(a)pyrene concentra-
tions, independently of the inoculation, possibly by a
combination of biological and non-biological effects.
These are promizing results and studies attempting to
identify the different mechanisms involved are currently
underway.
The high degradation values of phenanthrene and
anthracene in the non-inoculated soil indicated the
presence of PAH degraders. Experiments aimed at iso-
lating and identifying the microorganism/s responsible
for this degradation are planned.
Acknowledgements
The authors thank Professor Michael Goodfellow and
his group for their technical help in the identification of
Rhodococcus wrastislaviensis.
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